Anti-Mouse Ly-6G [Clone 1A8] — Purified in vivo PLATINUM™ Functional Grade

Anti-Mouse Ly-6G [Clone 1A8] — Purified in vivo PLATINUM™ Functional Grade

Product No.: L305

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Clone
1A8
Target
Ly-6G
Formats AvailableView All
Product Type
Monoclonal Antibody
Alternate Names
Lymphocyte Antigen 6 Complex
Isotype
Rat IgG2a
Applications
CyTOF®
,
Depletion
,
FC
,
IHC FF
,
in vivo
,
PhenoCycler®
,
WB

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Select Product Size

Data

Anti-Mouse Ly6G CyTOF™ Data
Clone 1A8 was used for neutrophil depletion in an acute zymosan-induced peritonitis model. 500µg of Clone 1A8 was dosed one day before the experiment. Zymosan was injected 3-hours prior to takedown.Clone 1A8 was used for neutrophil depletion in an acute zymosan-induced peritonitis model. 500µg of Clone 1A8 was dosed one day before the experiment. Zymosan was injected 3-hours prior to takedown.

Data generously provided by Dr. Nan Zhang Lab at Wistar Institute, Philadelphia, PA
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Antibody Details

Product Details

Reactive Species
Mouse
Host Species
Rat
Recommended Isotype Controls
Recommended Dilution Buffer
Immunogen
Ly-6G transfected EL-4J cell line.
Product Concentration
≥ 5.0 mg/ml
Endotoxin Level
<0.5 EU/mg as determined by the LAL method
Purity
≥98% monomer by analytical SEC
>95% by SDS Page
Formulation
This monoclonal antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.
Product Preparation
Functional grade preclinical antibodies are manufactured in an animal free facility using in vitro cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.
Pathogen Testing
To protect mouse colonies from infection by pathogens and to assure that experimental preclinical data is not affected by such pathogens, all of Leinco’s Purified Functional PLATINUM™ antibodies are tested and guaranteed to be negative for all pathogens in the IDEXX IMPACT I Mouse Profile.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Country of Origin
USA
Shipping
Next Day 2-8°C
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for this 1A8 antibody for staining cells in flow cytometry is ≤ 0.25 μg per 106 cells in a volume of 100 μl. Titration of the reagent is recommended for optimal performance for each application.
Additional Applications Reported In Literature ?
CyTOF®
Depletion
IHC FF
WB
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.

Description

Description

Specificity
Clone 1A8 recognizes an epitope on mouse Ly6G. Clone 1A8 does not cross react with Ly6C.
Background
Ly6G antibody, clone 1A8, recognizes lymphocyte antigen 6 complex locus G6D (Ly6G; also called Gr-1), a 21-25 kDa glycosylphosphatidylinositol (GPI)-anchored protein1. Ly6G belongs to the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily, characterized by a Ly6/uPAR (LU) domain-containing a three-fingered structural motif stabilized by disulfide bonds2. Ly6G is expressed by murine neutrophils regardless of location and activation1,4,5. Eosinophils may also express low levels of Ly6G5. There is no human ortholog for Ly6G; however, a structurally related L76/uPAR protein, CD177 (also known as HNA-2a, NB1, or PRV-1) is expressed in human neutrophils and is implicated in neutropenia6. Although the exact function and ligand of Ly6G remain unknown, Ly6G ligation may impair neutrophil migration to sites of inflammation via a β2-integrin-dependent mechanism7.
Antigen Distribution
Ly6G is expressed by neutrophils.
PubMed
NCBI Gene Bank ID
Research Area
Immunology
.
Innate Immunity

Leinco Antibody Advisor

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Clone 1A8 is most commonly used in vivo in mice for neutrophil depletion studies. This approach enables precise investigation of neutrophil function in diverse biological contexts, including infectious diseases, cancer, inflammation, wound healing, and autoimmunity.

Essential usage details:

  • Neutrophil depletion: 1A8 specifically targets mouse Ly6G, a surface marker exclusive to neutrophils, avoiding off-target depletion of other cell types (unlike clone RB6-8C5, which also affects monocytes and some T cells).
  • Applications across research areas: The antibody is employed to study neutrophil contributions to disease progression, immune defense, tumor microenvironments, and resolution of inflammation.
  • Tissue-specific depletion: 1A8 effectively removes neutrophils not only from peripheral blood but also from tissues such as tumors and the liver.
  • Experimental protocols: In vivo dosing typically ranges from 100–250 μg per mouse, administered intraperitoneally every 3 days, with the duration and frequency adjusted per model demands.

Additional in vivo applications:

  • Cancer immunotherapy studies: Used to assess the role of neutrophils in tumor growth, metastasis, and interactions with other immune cells.
  • Infectious and inflammatory disease models: Enables evaluation of neutrophil-mediated host protection vs. tissue damage.

Other laboratory uses (primarily in vitro or ex vivo, but often ancillary to in vivo work):

  • Flow cytometry: Verification of depletion and monitoring of cell populations.
  • Immunohistochemistry (IHC) and immunofluorescence (IF): Analysis of neutrophil presence and distribution in tissues post-depletion.

Summary Table: Common In Vivo Applications of Clone 1A8 in Mice

ApplicationDetails
Neutrophil DepletionSpecific removal of Ly6G+ neutrophils to study their in vivo functions
Infectious DiseaseDissecting neutrophil roles in pathogen defense and immunopathology
Cancer ModelsAssessing neutrophil involvement in tumor progression and immunity
Inflammation/AutoimmunityInvestigating contribution to acute and chronic inflammatory responses
Wound HealingEvaluating neutrophil effects on tissue repair/regeneration

Clone 1A8’s specificity and versatility have established it as the gold standard for in vivo neutrophil depletion in mouse immunology and disease modeling research.

The antibody 1A8 is most commonly used to target Ly6G for neutrophil depletion in mouse models. In the literature, it is frequently combined with the following antibodies and proteins to delineate immune cell populations or clarify cellular mechanisms:

  • CD11b: Identifies myeloid cells, including neutrophils and monocytes. The combination of Ly6G (1A8) and CD11b is a standard approach to confidently gate and analyze neutrophils in flow cytometry and immunophenotyping.
  • Ly6C: Differentiates between neutrophils and inflammatory monocytes; Ly6G and Ly6C are often used in parallel to distinguish these subsets since 1A8 is specific for Ly6G (neutrophils), while RB6-8C5 cross-reacts with both antigens.
  • CD45: Pan-leukocyte marker, useful for identifying total leukocyte populations in combination with more specific markers like Ly6G.
  • F4/80: Macrophage marker, helpful for excluding or profiling monocyte/macrophage populations when examining neutrophil-specific effects.
  • GR-1 (clone RB6-8C5): Recognizes both Ly6G and Ly6C and is used for comparison studies on specificity and depletion efficacy, but lacks the neutrophil specificity of 1A8.
  • CD3e: T cell marker, often included in panels to exclude lymphoid contamination or analyze overall immune landscape.
  • NK1.1: Natural killer (NK) cell marker, used in broader immunophenotyping panels.
  • B220/CD45R: B cell marker, similarly included for comprehensive leukocyte profiling.

In practical terms, an example multicolor flow cytometry panel to analyze myeloid and lymphoid subsets might include:

MarkerCell Population
Ly6G (1A8)Neutrophils
CD11bMyeloid lineage
Ly6CMonocytes (with Ly6G-)
CD45All leukocytes
F4/80Macrophages
CD3eT cells
NK1.1NK cells
B220/CD45RB cells

Summary: The most common antibodies or proteins used with 1A8 are those enabling precise leukocyte subset identification—CD11b, Ly6C, CD45, F4/80, alongside lineage markers like CD3e, NK1.1, and B220/CD45R. These combinations are used to characterize, separate, or deplete neutrophils within the broader context of the immune system.

Clone 1A8 is widely cited in scientific literature as a highly specific tool for depleting neutrophils in mice, allowing researchers to uncover the precise roles of neutrophils in various immune responses and disease models.

Key findings from 1A8-related citations include:

  • Neutrophil-specific depletion: 1A8 antibody targets the Ly6G protein, which is expressed exclusively on neutrophils, enabling researchers to deplete only neutrophils without affecting other cell types such as monocytes or eosinophils.

  • Immune role of neutrophils: Studies using clone 1A8 have demonstrated previously unknown roles for neutrophils in immunity, such as their contribution to bacterial clearance in Listeria monocytogenes infection, mediated through TNF-α pathways. Neutrophils were also found to be crucial in responding to acute inflammatory stimuli like zymosan-induced peritonitis.

  • Disease models and pathogen responses: Depletion of neutrophils with 1A8 has revealed that loss of neutrophil function can increase susceptibility and mortality in infectious disease models (e.g., bacterial and viral infections), highlighting their protective roles. For example, neutrophil depletion increased mortality in B6 mice infected with influenza, showing their essential function in early host defense.

  • Protocol optimization: The design and isotype of the antibody impacts depletion efficiency and duration. A murinized version of 1A8 (mouse-Ly6G IgG2a) enabled long-term and near-complete neutrophil depletion (>90%) in peripheral blood of several mouse strains, maintaining depletion for up to four weeks without anti-rat antibody development.

  • Inflammation and tissue injury: 1A8-targeted neutrophil depletion demonstrated that neutrophils can participate in acute inflammatory injury but might also promote pathological inflammation in chronic or autoimmune settings.

  • Methodological standards: 1A8 is recommended for both flow cytometric analysis and in vivo depletion studies, with established dosing protocols to optimize neutrophil-specific responses; these protocols emphasize the consistency required for model, strain, and experimental endpoint.

Summary Table—Key Findings from 1A8 Citations

Research AreaClone 1A8 Contribution
Neutrophil DepletionHighly specific; spares other immune cells
Infection ModelsReveals neutrophil role in pathogen clearance & mortality
Inflammation & InjuryDifferentiates neutrophil-mediated pathology from protective effects
Protocol OptimizationMurinized IgG2a variant provides long-term, efficient depletion
Experimental UsageStandard for immunological studies requiring neutrophil-specific analysis

Collectively, literature citations of clone 1A8 have transformed the understanding of neutrophil biology by affording researchers reliable, cell-type specific depletion in mice, thereby clarifying both beneficial and pathogenic roles of neutrophils in immunity and disease.

Dosing regimens for clone 1A8 (anti-Ly6G)—used for neutrophil depletion—typically range from 100–250 μg per mouse via intraperitoneal (i.p.) injection every 3 days or three times per week, but these parameters vary significantly depending on mouse strain, age, and disease model.

Key factors and variations:

  • Standard regimen: 100–250 μg per mouse, i.p., every three days or 3× per week, effective for neutrophil depletion in young C57BL/6 or BALB/c mice.
  • Mouse strain and age: Older mice (e.g., 24-week-old C57BL/6J) may show reduced or absent depletion at standard doses; efficacy must be experimentally verified. Some strains or conditions require protocol adjustments or higher doses.
  • Route of administration: While i.p. is most common, some studies use intravenous (i.v.) injection (e.g., 100 μg i.v. per dose ×2 for specific depletion), which may affect tissue targeting and depletion efficiency.
  • Disease model: In tumor, infection, or inflammation models, the same 100–250 μg i.p. regime is typically used, but depletion efficiency differs depending on disease context and neutrophil maturation. Some models may require adjunctive therapies or alternative strategies, particularly where depletion is less effective (e.g., certain infections or in the tumor microenvironment with immature neutrophils).
  • Compared to RB6-8C5: 1A8 is often dosed at roughly double the amount needed for the less specific RB6-8C5 antibody, as 1A8 is more selective but less efficient for depletion.
  • Body weight-based dosing: Some protocols use 7.5–20 mg/kg as an alternative to per-mouse dosing, emphasizing further adjustment for mouse size and age.
  • Long-term versus acute studies: Repeated dosing is necessary to maintain sustained neutropenia due to rapid neutrophil recovery post-depletion. Schedules remain about 3× per week, but the total duration and monitoring must be tailored to the study.
  • Verification of depletion: Regular flow cytometry or blood counts are critical to confirm actual neutrophil depletion in each experimental cohort, considering inter-animal and inter-strain variability.
  • Model-specific considerations: Some models, such as immunotherapy-treated tumors, may show more efficient depletion, while others (e.g., influenza-induced lung injury) can reveal persistence of neutrophil-like cells even after 1A8 treatment due to atypical Ly6G+ populations.

In summary, the optimal dosing regimen of clone 1A8 is highly context-dependent, with mouse strain, age, disease model, and experimental endpoint all requiring careful consideration and experimental validation. The standard starting point is 100–250 μg per mouse, i.p., three times weekly, but dose and schedule should be empirically optimized per model.

References & Citations

1. Fleming TJ, at al. (1993) J Immunol. 151(5):2399-408
2. Tsetlin VI. (2015) Trends Pharmacol Sci. 36(2):109-23
3. Daley JM, et al. (2008) J Leukoc Biol. 83(1):64-70
4. Lee PY, et al. (2013) J Leukoc Biol. 94(4):585-594
5. Percopo CM, et al. (2017) J Leukoc Biol. 101(1):321-328.
6. Stroncek DF. (2007) Curr Opin Hematol. 14(6):688-93
7. Wang JX, et al. (2012) Blood. 120(7):1489-1498
8. Tzetzo, S. L., Kramer, E. D., Mohammadpour, H., Kim, M., Rosario, S. R., Yu, H., Dolan, M., Oturkar, C. C., Morreale, B., Bogner, P. N., Stablewski, A., Benavides, F., Brackett, C. M., Ebos, J. M., Das, G. M., Opyrchal, M., Nemeth, M. J., Evans, S. S., & Abrams, S. I. (2024). Downregulation of IRF8 in alveolar macrophages by G-CSF promotes metastatic tumor progression. iScience, 109187. https://doi.org/10.1016/j.isci.2024.109187
CyTOF®
Depletion
Flow Cytometry
IHC FF
in vivo Protocol
PhenoCycler®
General Western Blot Protocol

Certificate of Analysis

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